Institute of Biochemistry, Graz University of Technology, Austria.
Plant Biotechnology, Faculty of Biology, University of Freiburg, Germany.
FEBS J. 2018 May;285(10):1923-1943. doi: 10.1111/febs.14458. Epub 2018 Apr 19.
The berberine bridge enzyme from the California poppy Eschscholzia californica (EcBBE) catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine, that is, the formation of the berberine bridge in the biosynthesis of benzylisoquinoline alkaloids. Interestingly, a large number of BBE-like genes have been identified in plants that lack alkaloid biosynthesis. This finding raised the question of the primordial role of BBE in the plant kingdom, which prompted us to investigate the closest relative of EcBBE in Physcomitrella patens (PpBBE1), the most basal plant harboring a BBE-like gene. Here, we report the biochemical, structural, and in vivo characterization of PpBBE1. Our studies revealed that PpBBE1 is structurally and biochemically very similar to EcBBE. In contrast to EcBBE, we found that PpBBE1 catalyzes the oxidation of the disaccharide cellobiose to the corresponding lactone, that is, PpBBE1 is a cellobiose oxidase. The enzymatic reaction mechanism was characterized by a structure-guided mutagenesis approach that enabled us to assign a catalytic role to amino acid residues in the active site of PpBBE1. In vivo experiments revealed the highest level of PpBBE1 expression in chloronema, the earliest stage of the plant's life cycle, where carbon metabolism is strongly upregulated. It was also shown that the enzyme is secreted to the extracellular space, where it may be involved in later steps of cellulose degradation, thereby allowing the moss to make use of cellulose for energy production. Overall, our results suggest that the primordial role of BBE-like enzymes in plants revolved around primary metabolic reactions in carbohydrate utilization.
Structural data are available in the PDB under the accession numbers 6EO4 and 6EO5.
加利福尼亚罂粟（Eschscholzia californica）中的小檗碱桥酶（EcBBE）催化（S）-延胡索乙素的氧化环化生成（S）-白屈菜红碱，即在苯并异喹啉生物碱生物合成中形成小檗碱桥。有趣的是，在缺乏生物碱生物合成的植物中已经鉴定出大量的 BBE 样基因。这一发现提出了 BBE 在植物界中的原始作用的问题，这促使我们研究Physcomitrella patens 中 EcBBE 的最接近的亲缘物（PpBBE1），这是最基础的植物，含有 BBE 样基因。在这里，我们报告了 PpBBE1 的生化、结构和体内特性。我们的研究表明，PpBBE1 在结构和生化上与 EcBBE 非常相似。与 EcBBE 不同的是，我们发现 PpBBE1 催化二糖纤维二糖氧化生成相应的内酯，也就是说，PpBBE1 是一种纤维二糖氧化酶。通过结构导向的诱变方法对酶的反应机制进行了表征，使我们能够确定 PpBBE1 活性位点中氨基酸残基的催化作用。体内实验表明，PpBBE1 在植物生命周期的最早阶段——绿丝体中表达水平最高，此时碳代谢强烈上调。还表明该酶被分泌到细胞外空间，在那里它可能参与纤维素降解的后续步骤，从而使苔藓能够利用纤维素进行能量生产。总的来说，我们的结果表明，BBE 样酶在植物中的原始作用是围绕碳水化合物利用中的初级代谢反应展开的。
结构数据可在 PDB 中以 6EO4 和 6EO5 的访问号获得。
